The Green Leafy Vegetable Psophocarpus Scandens as Putative...

Pharmaceutical Science and Technology 2018; 2(2): 7-13

http://www.sciencepublishinggroup.com/j/pst

doi: 10.11648/j.pst.20180202.11

ISSN: 2640-4532 (Print); ISSN: 2640-4540 (Online)

The Green Leafy Vegetable Psophocarpus Scandens as Putative Source of Nutraceuticals in Sickle Cell Disease: The Scientific-Based Evidences

Koto-te-Nyiwa Ngbolua1, 2, *

, Nathanael Nieto Kongobi3, Clément Liyongo Inkoto

1,

Gédeon Ngiala Bongo2, Colette Masengo Ashande

2, Clément Mutunda Mbadiko

1,

Clarisse Mawi Falanga1, Benjamin Zoawe Gbolo

1, 2, Pius Tshimankinda Mpiana

4

1Department of Biology, University of Kinshasa, Kinshasa, Democratic Republic of the Congo 2Department of Environmental Sciences, University of Gbadolite, Gbadolite, Democratic Republic of the Congo 3Section of Pure Science, Medical Techniques High School of Yakoma, Yakoma, Democratic Republic of the Congo 4Department of Chemistry, University of Kinshasa, Kinshasa, Democratic Republic of the Congo

Email address:

*Corresponding author

To cite this article: Koto-te-Nyiwa Ngbolua, Nathanael Nieto Kongobi, Clément Liyongo Inkoto, Gédeon Ngiala Bongo, Colette Masengo Ashande, Clément

Mutunda Mbadiko, Clarisse Mawi Falanga, Benjamin Zoawe Gbolo, Pius Tshimankinda Mpiana. The Green Leafy Vegetable Psophocarpus

scandens as Putative Source of Nutraceuticals in Sickle Cell Disease: The Scientific-Based Evidences. Pharmaceutical Science and

Technology. Vol. 2, No. 2, 2018, pp. 7-13. doi: 10.11648/j.pst.20180202.11

Received: October 13, 2018; Accepted: October 26, 2018; Published: November 16, 2018

Abstract: In the Democratic Republic of the Congo (DRC), medicinal plants represent the main product for both urban and

rural populations for their health care needs due to the high costs of conventional medicine. These plant species contain

bioactive compounds also called phytochemicals that are capable of modulating metabolic processes and resulting in the

promotion of better health. Some of these plants act therefore as functional foods and could serve as sources of nutraceuticals.

Psophocarpus scandens, an unconventional green leafy vegetable, is well known to have antioxidant activity which is one of

the modes of action of Sickle cell disease (SCD) drugs. To justify this study, it was hypothesized that P. scandens possess

antisickling properties. The aim of the present study was to evaluate the antisickling activity of organic acids rich extract from

P. scandens using in vitro biological experiments (Emmel, Itano and hemolysis tests). The results revealed that P. scadens have

antisickling properties in vitro (normalization rate: 80%). This bioactivity is expressed as the re-appearance of the normal and

classical biconcave form of red blood cells (RBCs: from 0.00±0.00 to 3.55±0.28 µm) in hypoxic conditions by reducing the

perimeter of sickle RBCs (from 35.26±1.21 to 19.80±1.15 µm) and increasing their surface (from 21.41±1.84 to 34.10±1.76

µm2) (p<0.05). The bioactivity displayed could be due to triterpenoic acids which are able to reduce both the rate of hemolysis

(hemolysis inhibition ˃50%) and the aggregation of deoxy-Hb S (hemoglobin S polymerization inhibition rate ˃80%) as

experimentally demonstrated. Literature search revealed that this green leafy vegetable contains polyphenolic compounds

(glucosylated flavonoids and phenolic acids) with antioxidant properties and all essential amino-acids. Thus, the daily

consumption of P. scandens fortified with the leaves powder of Moringa oleirera could considerably reduce in vivo oxidative

stress and hemolysis associated with clinical manifestation of SCD. Based on current knowledge this is a first report on the

antisickling activity of P. scandens. It is desirable that the bio-guided fractionation of organic acids extract could be carried out

in order to isolate and elucidate the structure of bioactive pure compound(s).

Keywords: Sickle Cell Disease, Functional Foods, Nutraceuticals, Psophocarpus Scandens, Evidences-Based Medicine

1. Introduction Tropical medicinal plant species are known for their

richness in biologically active secondary metabolites of

8 Koto-te-Nyiwa Ngbolua et al.: The Green Leafy Vegetable Psophocarpus scandens as Putative Source of

Nutraceuticals in Sickle Cell Disease: The Scientific-Based Evidences

therapeutic relevance [1]. The principal advantages claimed

for therapeutic uses of botanicals against various ailments are

their safety besides being economical, effective and their

easy availability. According to these advantages, medicinal

plants are widely used by traditional healers in their day to

day practice [1, 2].

In the Democratic Republic of the Congo (DRC),

medicinal plants represent the main product for both urban

and rural populations for their health care needs due to the

high costs of conventional medicine [3-5]. These plant

species contain bioactive compounds also called

phytochemicals that are capable of modulating metabolic

processes and resulting in the promotion of better health.

Some of these plants act therefore as functional foods and

could serve as sources of nutraceuticals [6].

The use of edible medicinal plants is an interesting

approach since these plants can be integrated into the daily

diet of patients suffering from Sickle cell disease (SCD). Ten

medicinal foods namely Cajanus cajan, Sorghum bicolor,

Ipomea batatas, Moringa oleifera, Adansania digitata,

Ocimum basilicum, Vigna unguiculata, Gnetum africana,

Grewia coriacea and Uvariodendron molundense were

scientifically validated as having antisickling activity in vitro.

This activity is mainly due to anthocyanins and organic acids

and their derivatives [7-9].

An interesting study on the Congolese traditional foods as

putative sources of antioxidants with health benefits in

Konzo [6, 10], a toxico-nutritional neurological disease

associated with oxidative damage due to free radicals (like

SCD), revealed that the unconventional green leafy vegetable

Psophocarpus scandens have antioxidant activity (one of the

modes of action of antisickling drugs). It can therefore, be

hypothesized that P. scandens possess antisickling properties,

thus justifying this research work. The aim of the present

study was to extract and evaluate the antisickling activity of

organic acids rich extract from P. scandens. Indeed, SCD is a

genetic disease due to the presence of hemoglobin S (Hb S)

in the blood which could in hypoxia conduct to the formation

of tactoids, the leading cause of erythrocyte sickling that

plays a key role in the pathophysiology of SCD like vaso-

occlusion (due to the loss of membrane elasticity) and

hemolytic anemia. There is strong evidence that the

polymerization of deoxy Hb S substantially lowers its oxygen

affinity. In the sickling state, the erythrocytes of SCD patients

contain 3-4 times more than the normal concentration of the

calcium ions as a result of ATP depletion. Since there are

little or no endocytic vesicles for calcium ion storage, most

of the calcium ions are bound to membrane proteins, thereby

resulting in dehydration and hemochrome formation with a

resulting loss of erythrocytes deformability, cell-to-cell

adherence and free radicals production. However, recent

findings indicate that plant-derived antisickling agents may

directly inhibit polymerization of hemoglobin, free radicals

production or modify membrane stability. The membrane

stability is evaluated in this study by measuring the

erythrocytes hemolysis [11, 12].

2. Materials and Methods

2.1. Plant Material Collection and Authentication

The tested plant materials used in this study were collected

in DRC in March 2013 and were authenticated at INERA

(Institut National d’Etudes et Recherches Agronomiques).

Voucher specimen is kept in INERA Herbarium (Faculty of

Science, University of Kinshasa).

2.2. Extraction and Phytochemical Screening

The dried and powdered plant material (10 g) was

repeatedly extracted by cold percolation with 95% ethanol

(EtOH) and water (100 mL x 2) for 48 hours. Chemical

screening was performed on the aqueous and organic extracts

to investigate the presence of alkaloids, saponins, total

polyphenols, flavonoids, tannins, anthocyanins, leuco-

anthocyanins, quinones and triterpenoids according to

standard protocol [13].

2.2.1. Detection of Phenols (Ferric Chloride Test)

Extracts were treated with 3-4 drops of ferric chloride

solution. Formation of bluish black colour indicates the

presence of phenols.

2.2.2. Detection of Flavonoids

The ethanol extract (5 ml) was added to a concentrated

sulphuric acid (1 ml) and 0.5 g of Mg. A pink or red

coloration that disappear on standing (3 min) indicates the

presence of flavonoids.

2.2.3. Detection of Anthocyanins

The presence of anthocyanins is revealed by a color

change as a function of pH due to titration of the acidic

aqueous solution with a solution of NaOH. If the solution

turns a red color, the pH is less than 3, if against a blue color;

the pH is between 4 and 6.

2.2.4. Detection of Tannins

Two methods were used to test for tannins. First, about 1

ml of the ethanol extract was added in 2 ml of water in a test

tube. 2 to 3 drops of diluted ferric chloride solution was

added and observed for green to blue-green (cathechic

tannins) or a blue-black (gallic tannins) coloration. Second, 2

ml of the aqueous extract was added to 2 ml of water, a 1 to 2

drops of diluted ferric chloride solution was added. A dark

green or blue green coloration indicates the presence of

tannins.

2.2.5. Detection of Leucoanthocyanins

To 2 ml of aqueous extract was added few drop of Shinoda

regeant in a test tube and then boiled. A red or purple

coloration in the supernatant indicates the presence of

leucoanthocyanins.

2.2.6. Detection of Saponins

To 1 ml of aqueous extract was added few volume of

Pharmaceutical Science and Technology 2018; 2(2): 7-13 9

distilled water in a test tube. The solution was shaken

vigorously and observed for a stable persistent froth for 20

min.

2.2.7. Detection of Alkaloids

Five ml of the extract was added to 2 ml of HCl. To this

acidic medium, 1 ml of Dragendroff’s reagent was added. An

orange or red precipitate produced immediately indicates the

presence of alkaloids.

2.2.8. Detection of Free Quinones

To 1 ml of organic extract was added few drops of

Borntrager reagent (NaOH 10% ou NH4OH 10%) in a test

tube. The solution was and then shaken vigorously. A sharp

red or orange coloration indicates the presence of free

quinones.

2.2.9. Detection of Triterpenoids

Ten (10) mg of the extract was dissolved in 1 ml of

chloroform; 1 ml of acetic anhydride was added following

the addition of 2 ml of Conc. H2SO4. The formation of

reddish violet colour indicates the presence of triterpenoids.

2.3. Organic/Triterpenic Acids Extraction

The powdered material (40 g) were macerated with 100

mL of dichloromethane-methanol-NH4OH (100:1:1; v/v/v)

and then percolated with 300 mL of the same solvent mixture

at room temperature. The extract was concentrated under

reduced pressure until 100 mL (pH 10). The resulting

solution was then mixed with 5% citric acid (v/v) to

precipitate organic/triterpenic acids [14]. Fractions were

filtered and concentrated to dryness under reduced pressure

using a rotary evaporator.

2.4. In Vitro Antisickling Activities

2.4.1. Blood Samples Collection

Blood samples used to assess the antisickling activity were

taken from known SCD patients attending the “Centre de

Médecine Mixte et d’Anémie SS” located in Kinshasa,

Democratic Republic of the Congo. None of the patients had

been transfused recently with Hb AA blood and all

antisickling experiments were carried out with freshly

collected blood. In order to confirm their SS nature, the

above-mentioned blood samples were first characterized by

Hemoglobin electrophoresis on cellulose acetate gel to

confirm their status and were then stored at +4 °C in a

refrigerator.

An informed consent was obtained from all the patients

participating in the study and all the research procedures

have received the approval of Department of Biology Ethics

Committee.

2.4.2. Emmel Test

An aliquot of Hb S-blood was diluted with 150 mM

phosphate buffered saline (NaH2PO4 30 mM, Na2HPO4 120

mM, NaCl 150 mM) and mixed with an equivalent volume of

2% sodium metabisulfite. A drop from the mixture was

spotted on a microscope slide in the presence or absence of

plant extracts and covered with a cover slip.

Paraffin was applied to seal the edges of the cover

completely to exclude air (Hypoxia). Duplicate analyses were

run for each extract. The red blood cells (RBCs) were

analyzed by a computer assisted image analysis software

(Motic Images 2000, version 1.3; Motic China Group Co

LTD).

2.4.3. Anti-Hemolytic Assay

Erythrocytes (RBCs) were washed twice in physiological

saline (NaCl 0,9%, 1:5 v/v) by centrifugation at 3000 rpm for

10 min, re-suspended in phosphate buffer (150 mM, pH 7,4)

containing 2% sodium metabisulfite and incubated in the

absence (control) or presence of ethanolic extract of P.

scandens (50 µg/mL)/organic acids extract of P. scandens (50

µg/ml of basified NaCl 0,9%) at 37°C for 60 min. At fixed

time points, aliquots of the blood samples were removed and

centrifuged at 3000 rpm at ambient temperature for 5 min.

The absorbance of the supernatant was measured at 540 nm

and was expressed as the degree of haemolysis. The rate of

hemolysis inhibition (% HI) versus time was calculated from

the absorbance of sickle erythrocyte (SS RBCs) suspension

by the relation:

%HI = 100 X [Absorbance of untreated SS RBCs suspension

Absorbance of treated SS RBCs suspension2a]

The zero time corresponds to 30 min after pre-incubation

of SS RBCs with plant extracts (treated SS RBCs) or NaCl

0,9% (untreated SS RBCs) in hypoxic conditions [11, 12].

2.4.4. Anti-Gelling Assay (Itano test)

Erythrocytes were washed twice in physiological saline

solution (NaCl 0.9%) by centrifugation at 3000 rpm for 10

min, re-suspended in hypotonic medium. After that, the

hemolysate of RBCs was centrifuged and an equivalent

volume of 2% metabisulfite was added to supernatant. It was

than incubated at ambient temperature for 45 min. At fixed

time points aliquots (50 µL) of the 2% sodium metabisulfite

pre-treated hemolysate were diluted with 500 µL of

phosphate buffer (pH 7.5) containing (NH2)2SO4 30%,

Saponine 1% and K2HPO4 1.2%. 50 µL of ethanolic or

organic acids extract of P. scandens (50 µg/mL) were added

to the test sample, mixed and incubated for 10 min. The

equivalent volume of phosphate buffered saline (PBS) was

added to the control sample instead of the drug. At

predetermined time intervals aliquots of test or control

samples were removed and centrifuged at 3500 rpm at

ambient temperature for 5 min. The absorbance of the

supernatant was measured at 700 nm. The solubility of the

deoxygenated sickle cell hemoglobin was expressed as the

decrease of the optical density at 700 nm (anti-gelling effect).

The zero time corresponds to 30 min after pre-incubation of

Hb S with plant extract (treated Hb S) or NaCl 0,9%

(untreated HbS) in hypoxic conditions [11, 12].

2.5. Literature Search



A deep literature search was carried out in order to obtain

information about the traditional uses, microscopic features,

nutritional value, phytochemistry and bioactivities of P.

scandens from various electronic databases namely PubMed,

PubMed Central, Science Direct and Google scholar as

previously reported [1, 4].

3. Results and Discussion

3.1. Laboratory Assays Results

The chemical screening performed on the leaves of P.

scandens revealed the presence of alkaloids, saponins, total

polyphenols, flavonoids, tannins, anthocyanins, triterpenoids

and free quinones.

Phenolic compounds such as anthocyanins [7, 11, 12, 14],

rosmarinic acid [15] and lunularic acid [16] and triterpenes

like betulinic, maslinic, oleanolic [17] and ursolic acid [18]

were reported to display antisickling activity in vitro.

The figure 1 gives the morphology of untreated sickle

erythrocytes (a) and SS RBCs treated with 50 µg/mL of P.

scandens organic acids extract (b).

[A]

[B]

Figure 1. Morphology of untreated sickle erythrocytes [A] or SS RBCs

treated with 50 µg/mL of P. scandens organic acids extract [B] (X500),

[NaCl 0.9%; Na2S2O5 2%].

Figure 1a reveals that the control sample contains in

majority sickle-shaped erythrocytes, confirming the SS

homozygous status of the used blood. Mixed together with

organic acid extract (Fig. 1b); the majority of erythrocytes are

reversed normal-shape as revealed in the table 1. This indicates

that organic acids extract of P. scadens have antisickling

properties. The bioactivity displayed could be due to

triterpenoic acids as previously reported [14].

Table 1. Average values of radius, perimeter and surface of erythrocytes

before and after treatment with P. scandens organic acids rich extract.

Measured parameters Untreated SS

RBCs

SS RBCs

(+P. scandens OA extract)

Radius (µm) 0.00±0.00 3.55±0.28

Perimeter (µm) 35.26±1.21 19.80±1.15

Surface (µm2) 21.41±1.84 34.10±1.76

(Legend: OA, organic acids)

As it can be seen in table 1, the used computer software

package/program did not give the average radius for

drepanocytes, as sickled cells of untreated SS blood are not

circular. The average radius appeared after treatment of SS

RBCs by P. scandens organic acids rich extract (50 µg/mL),

conduct into the re-appearance of the normal and classical

biconcave form of RBCs by reducing the perimeter of sickle

RBCs and increasing their surface (p<0.05), thus confirming

the antisickling effect of P. scandens.

Figure 2 shows the dose dependent antisickling activity of

triterpenic (triterpenoic) acids rich/organic acids extract from

P. scadens.

Figure 2. Dose-dependent normalization rate of sickled erythrocytes (NaCl

0.9%; Na2S2O5 2%) (The curve was fitted with the help of Origin Pro 8.5

package software using B-spline: Fill area under the curve and inclusive

broken by missing values).

It can deduce from the figure 2 that the normalization rate

of sickled cells in hypoxic conditions increases with the

extract dose. At the dose of 50 µg/mL the rates of

normalization were 80%. Thus, the antisickling activity of

the tested plant extract is dose dependent. It is therefore

suggested that bioactive extract could exert it


pharmacological effect by various mechanisms including the

inhibition of free radicals formation, and the inhibition of

erythrocyte hemolysis (figure 3) and the inhibition of

hemoglobin polymerization (figure 4) as previously reported

[11, 12, 14]. Thus, the daily consumption of P. scandens

fortified with the leaves powder of Moringa oleirera could

considerably reduce oxidative stress and hemolysis

associated with clinical manifestation of SCD.

Figure 3. In vitro effect of ethanolic extract (EtOH) and organic acids

extract from P. scandens on the hemolysis of Sickle red blood cells (RBCs)

(NaCl 0.9%; Na2S2O5 2%).

Figure 3 reveals that after 60 min of incubation, the rate of

RBCs hemolysis inhibition of the extracts of P. scandens is

greater than 50%. The best result was obtained with organic

acids extract.

Figure 4. In vitro effect of ethanolic and organic acids extracts of P.

scandens on the aggregation of deoxy-Hb S (Phosphate buffered saline 150

mM; pH 7.4; 2% Na2S2O5).

The figure 4 indicates that, in the untreated hemoglobin

aqueous solution (control) the absorbance at wave length of

700 nm increase with the time as a result of the loss of

hemoglobin solubility in hypoxic conditions created by 2%

Sodium metabisulfite. However, after addition of plant

extract, the absorbance decreases in the course of time. These

results indicate that ethanolic and organic acids extracts of P.

scandens inhibit the polymerization of hemoglobin S into

tactoids (anti-gelling effect). The best result was obtained

with organic acids extract.

3.2. Literature Search Results

P. scandens is a traditional food commonly known as

kikalakasa in DRC where the plant is consumed all over the

country. According to Mangala et al. [6], phytochemical studies

(TLC and HPL-DAD) of extracts from P. scandens revealed the

presence of polyphenolic compounds (glucosylated flavonoids

and phenolic acids). P. scandens contains also flavonoids (40, 78

± 1, 1l mg QE/g DW), tannins (102, 56 ± 0, 08 CE/g DW). The

extracts from this plant species displayed moderate antioxidant

activity in vitro (radical ATBS, IC50: 53, 2 ± 0, 97 µg/mL;

radical DPPH, IC50: 62, 81 ± 0, 97 µg/mL).

Study on the botanical microscopic characters of the leaves

powder of P. scandens revealed abundant prisms of calcium

oxalate crystals, starch grains, fragments of lignified fibers,

diacytic stomata, circular epidermal cells, spiral vessels and

unicellular non glandular trichomes. These results indicate that

the studied unconventional green leafy vegetable could provide

benefits in protecting SCD patients against oxidative damage.

The assessment of the nutritional value of P. scandens

revealed that this unconventional green leafy vegetable contains

(in g/100 g FM): crude proteins (7, 03), fats (1, 28),

carbohydrates (5, 40), crude fibers (2, 21), total ash (1, 38). This

corresponds to the energy value of 61 kCal and the moisture was

evaluated to 82, 7%. Biochemical analysis revealed also the

presence of all essential amino-acids [19]. This tropical plant

species could putatively be used as functional food to prevent

erythrocytes sickling and oxidative damage associated with

SCD. In DRC, 12% of the children hospitalized in the hospital

are sickle cell disease patients and it is estimated that the annual

cost of the treatment is higher than 1,000 USD per patient, a cost

hard to bear for the majority of the population whose average

income is lower than 2 USD per day and who for the needs for

primary health care turns mainly to Traditional Medicine [9].

Thus, P. scandens can be integrated into the daily diet of SCD

patients for a sustainable solution.

4. Conclusion and Suggestions

The aim of the present study was to evaluate the

antisickling activity of organic acids rich extract of P.

scandens. The results revealed that P. scadens have

antisickling properties in vitro (normalization rate: 80%).

This bioactivity is expressed as the re-appearance of the

normal and classical biconcave form of RBCs in hypoxic

conditions by reducing the perimeter of sickle RBCs and

increasing their surface. The bioactivity displayed could be

due to triterpenoic acids which are able in vitro to reduce

both the rate of hemolysis and the aggregation of deoxy-Hb

S. Thus, the daily consumption of P. scandens fortified with



the leaves powder of Moringa oleirera could considerably

reduce in vivo oxidative stress and hemolysis associated with

clinical manifestation of SCD. Based on current knowledge

this is a first report on the antisickling activity of P. scandens.

It is desirable that the bio-guided fractionation of organic

acids extract could be carried out in order to isolate and

elucidate the structure of bioactive pure compound(s) using

the combination of chromatographic and spectroscopic

techniques (LC/MS/NMR, FT-IR).

Acknowledgements

The authors are indebted to The International Foundation for

Science (IFS, Stockholm, Swedish) for Research Grant No.

F/6129-1 offered to Clément Mbadiko Mutunda and the

“Académie de Recherche et d’Enseignement Spérieur” (ARES,

Belgium) for Research Grant offered to Benjamin Gbolo Zoawe.

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